This research in response to the PAR-17-045 ?Focused Technology Research and Development? proposes to develop a non-invasive, deep-tissue imaging technology with single-cell sensitivity based on ultrasound and photoacoustic imaging. Ultrasound imaging uses sound wave to provide anatomic information of tissue, and offers many desirable characteristics--fast, real-time imaging, low cost, deep tissue penetration, high spatial resolution and no exposure to ionizing radiation. However, the presence of significant speckle noise greatly compromises the imaging quality and resolution. Photoacoustic (PA) imaging uses non-ionizing laser pulse excitation to generate ultrasound emission that is detectable by ultrasound transducers. It combines the spectroscopic- based specificity and high-contrast of optical imaging at deep-tissue location (~ a few cms). However, blood and pigments generate high intrinsic background signals, which significantly limit the in vivo sensitivity in detecting molecular and cellular targets. This research will explore innovative engineering and nanotechnology to address these challenges to enable non-invasive, deep-tissue imaging technology with single-cell sensitivity. Aim 1 will develop nonlinear difference-frequency generation for ultrasound imaging to provide highly contrasted anatomic information. Aim 2 will develop photoswtichable photoacoustic nanoparticles to enable near-infrared photoswitchable photoacoustic imaging and provide single cell imaging sensitivity. Aim 3 will integrate ultrasound imaging with photoswitchable photoacoustic imaging to image tumor lymph nodes and demonstrate the synergy of Aim 1 and Aim 2 techniques in obtaining high-contrast anatomical and molecular information in deep tissue of living subjects. At the end of the 4 years period of funding, the research will produce an instrument prototype and novel nanoparticles that can be used for speckle-free acoustic (anatomic tissue information), photoswitchable photoacoustic imaging (single-cell molecular and cell information). It is expected that this new imaging technology will provide unprecedented opportunity in acquiring information on biological molecules in complex, native physiological settings and enable many fundamental biology discoveries.